The present invention relates to methods for anplifying nucleic acid sequences. In particular, the invention concerns methods for detecting the presence of a particular nucleic acid sequence with high sensitivity.
The detection of specific nucleic acid sequences is gaining rapid importance in a variety of fields, particularly in the field of medical diagnosis. Nucleic acid hybridization methods provide assays for detecting nucleic acid sequences of medical significance, such as DNA or RNA sequences indicative of genetic diseases, cancer, and bacterial and viral infections. Nucleic acid hybridization assays are based on the very specific base pairing that is found in hybrids of DNA and RNA. Base sequences of analytical interest appearing along a strand of nucleic acid can be detected very specifically and sensitively by observing the formation of hybrids in the presence of a probe nucleic acid known to comprise a base sequence that is complementary with the sequence of interest.
It is evident that for hybridization assays to attain their full analytical potential, methods for increasing the sensitivity of detection even further are needed. Considerable efforts have been applied to this aspect in recent years and a number of different approaches have been conceived and developed. Particularly promising are approaches based on the biochemical amplification of the target nucleic acid sequence or its complementary signal sequence. While detection systems each have their own sensitivity limits, biochemical systems have been developed which can make millions and millions of copies of the target or signal sequences thereby to extend the effective sensitivity limits of such detection systems by many orders of magnitude.
One such nucleic acid amplification method is that known as the polymerase chain reaction method, or PCR, which is described in U.S. Pat. Nos. 4,683,195 and 4,683,202. PCR employs a pair of specific oligonucleotide as primers for the two complementary strands of the double stranded form of a target sequence. The primers are chosen such that they form specific hybrids at the opposite 3' ends of the complementary target strands. Using a thermostable DNA polymerase, the primers are extended synthetically in correspondence with the target sequences. A thermal cycling process is required in order to form specific hybrids and, after extension, to denature the hybridized, extended strands for further primer hybridization and extension. Repeating the process several times results in a geometric amplification of the amount of the target sequences in the mixture.
A variation of PCR is the ligase chain reaction (LCR) described in European Patent Publication 320,308. This method requires at least four separate oligoprobes, two of which hybridize to opposite ends of the same target strand such that when they are hybridized to the target sequence their respective 3' and 5' ends are juxtaposed for ligation. The third and fourth probes hybridize with the first and second probes to form, upon ligation, fused probes which can be denatured and detected.
Another known amplification method is described in PCT Publication No. 88-10315 and will be referred to as the transcription amplification system or TAS. Similar methods are described in European Patent Publication No. 310,229 and PCT Publication No. 88-10315. As in PCR, TAS uses pairs of oligoprimers to hybridize with opposite ends of a desired target sequence. The primers are chosen such that the extension products, after either a single extension or multiple cycles as in PCR, comprise transcription promoter sites. In the presence of a suitable promoter specific polymerase and ribonucleoside triphosphates (rNTPs), the extension products are themselves further amplified by transcription.
The Q.beta. replicase (Q.beta.R) method described in PCT Publication No. 87-06270 uses a specific RNA probe which is capable of specific transcription by a replicase enzyme. The method has linear reaction kinetics and requires the design and synthesis of RNA probes with replicase initiation sites.
While all of these methods yield amplification of a target nucleic acid sequence, none are without complexities which are undesirable for the general and unsophisticated user. Many of the prior art methods require multiple incompatible steps that can be accomplished only by cumbersome manual procedures or complex and expensive instruments for automating the many manipulations required. Further, many require the preparation of multiple sophisticated reagents which limits the ready application of the methods to different target sequences.
Unrelated to the above pursuits, there have been studies of a variety of synthetic and naturally occurring DNA and RNA structures and their functions. One such structure is that known as the hairpin in which self-complementary regions in a single polynucleotide hybridize under suitable conditions to form looped structures whose shape resembles a common hairpin. Such hairpin structures are known to occur naturally in many organisms, particularly in RNA secondary structures, however, their functional role is at this point not well established. The physical chemistry of hairpin structures has been described --Cantor and Schimmel, Biophysical Chemistry, Part III, p. 1183, W. H. Freeman & Co. (San Francisco 1980).
The literature on this subject is incomplete and contradictory. For example, there are predictions that hairpins may provide a transcription termination signal--Jendrossek et al, J. Bacteriol. 170:5248 (1988) and Walker et al, Biochem. J. 224:799 (1984). Hairpin structures resembling known rho dependent transcription termination signals have been observed following the unc operon and glms of E. coli. On the other hand, palindromic sequences capable of forming stable hairpin forms have been found around the transcription initiation site of beta amyloid precursor gene--La Fauci et al, Biochem. Biophys. Res. Commun. 159:297 (1989).
The use of hairpin structures in the synthesis of DNA from oligonucleotides and in the labeling of oligonucleotides is proposed in European Patent Publication 292,802 and by Sriprakash and Hartas, Gene Anal. Techn. 6:29-32 (1989). In addition, Krupp and Soll, FEBS Letters 212:271 (1987) and "Nucleic Acid Probes", ed. Symons (CRC Press, Bacon Raton, Fla., 1989) pp. 21 & 22, describe the use of a hairpin structure to make labeled RNA transcripts from an M13 vector/T7 RNA polymerase system.